TCRExpress™ and SuperTCRExpress™ Technology



 
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T cells, TCR & CDR3

T cells belong to a group of cells called “lymphocytes” and play an important role for cell-mediated immunity. T cells can be distinguished from other lymphocytes by a special receptor on their cell surface, the so called T cell receptor (TCR). This is a molecule that is responsible for recognizing antigens, which are usually proteins or polysacharides, but may also be bacteria or viruses. The majority of T cell receptors are composed of an α-chain and a β-chain.

The TCR β-chain gene complex includes variable (V), diversity (D), joining (J) and constant (C) gene segments, and the expression of these gene segments determines its affiliation to a distinct TCR Vβ gene family. The blueprint of a TCR α-chain is similar to that of a β-chain, but it does not include a D gene segment.

The value of the T cells for the immune system comes from their diversity. For almost all possible antigen variants there is usually a T cell available with a TCR that will exactly fit to the antigen like a key to its lock, and can therefore be used to identify the antigen and trigger further immune response.

This T cell diversity is formed through hypervariable regions in the gene sequence of the cells. The region that shows most variability is the so called “complementary-determining region 3” (CDR3), and it includes parts of the V, D and J gene region in the β-chain (see Model 1), and V and J in the α-chain.

Model 1
 
Model 1: T cell receptor Vβ genes and their CDR3 region

The immune response through T cells depends on the number of T cell clones available that show specific receptors for the involved antigen. Therefore, the measurement of the number of different clones, as well as the number of cells of a specific clone, can give invaluable information regarding, for example, the preparedness of the body for an effective immune response, or the state of recovery from an attack. The assay of TCR repertoire clonality and diversity has been applied as a monitoring tool to detect the extend of viral, tumor or self-immune antigen induced T cell pathogenic clonotypes. Through immunization the expansion of specific physiologic clonotypes is initiated, and the number of cells with specific TCR’s will increase many times over the number of other clones. In this stage, the body has the best ability to resist the specific attacker it was immunized against. After I while, this effect is usually reduced and returns finally to the same stage as before the immunization. The assay also can be used to indicate T cell immune reconstitution following bone marrow transplantation, immunologic therapy, anti-tumor or anti-viral treatment in human subjects or animal models. Moreover, another important application of the TCR assay is to use it as a screening tool to screen TCR clones, such as CTL (cytotoxic T lymphocyte) clone, in the established monoclonal T cells.

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Current technology for TCR clonality assessment and its downfalls

At present, there are two main methods for measuring the diversity of the TCR repertoire. First, it can be assessed by the measurement of T cell activation using monoclonal antibody-based flow cytometry. Second, TCR repertoire clonality/diversity can be measured using PCR-based assay. The latter is a more precise assessment of TCR clonality/diversity in the CDR3 region. Its main principal is that clones within a Vα or Vβ family consist of amino acid (AA) chains with multiple lengths. The AA length does not have a one-to-one relationship to a specific clone, but all cells of one clone share the same AA length. The ability to group each family into length clusters gives the researcher the opportunity to analyze the TCR repertoire in a more detailed way than just by separating them into families alone. For example, naïve T cells leaving the thymus show an almost normal or Gaussian distribution of CDR3 size within individual gene families, while T cells in the peripheral T cell pools display clonal expansion or depletion due to specific antigen stimulation. However, the method still has several disadvantages. It requires access to a DNA analyzer, is time- and labor-intensive, and is subject to personal technical error, thus preventing its use as a rapid and routine assessment of patient samples from clinical trials. The quality of primers used and the diversity of end labeling sites also highly affect the accuracy of the assessment. Moreover, the lack of statistical and quantitative methods in the analysis of the spectratyping data also significantly reduces the usefulness of this technique. Until now, this technology has only been sporadically adopted by investigators and clinics.

Most of academic scientists and clinical investigators still lack a method to detect clonality or CDR3 size diversity of TCR repertoires reliably, rapidly, easily, and quantitatively following antigen driven specific immune responses in human and animal models.

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Our technology and products

Our scientific group has identified the problem as described above and successfully developed a series of novel assay kits that eliminates these problems or reduces them to a minimum. The kits can be used to determine rapidly, sensitively, reliably, and quantitatively clonalities, CDR3 diversity, and clone components of the TCR repertoire in human or mouse T lymphocytes and their subsets, such as CD4+, CD8+, CD4+CD45RA+ or RO+, CD8+CD45RA+ or RO+, CD4+CD25+, and CD4+CD25+ T cell subsets.

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T cell receptor clonality detecting kits

The novel TCRExpress clonality detecting kit can be used to detect quickly, sensitively, and reproducibly clonality and diversity in the CDR3 region of the TCR Vβ repertoire in human or mouse T lymphocytes and their subsets. This assay system consists of V-D-Jβ specific PCR amplifications and a gel - based assay (using a standard horizontal gel tray). It does not require a more costly DNA analysis for reliable results. The assay can be completed in a few hours, including PCR running time.

Figure 1
Figure 1: Clonalities of cord blood, Jurkat and HAV vaccinated CD4+CD45RA+ and CD4+CD45RO+ T cells detected using the TCRExpress assay system. In panel A, the cord blood T cells show smear (polyclonal repertoire) with no defined bands, while Jurkat T cells display a single defined band (monoclonal repertoire) in the same Vβ 8 gene family. Panel B shows that clonal expansion in the Vβ 7 and 20 families are only seen in the HAV specific subset CD4+CD45RA+ post immunization, but not pre immunization and also not in the T cell subset CD4+CD45RO+ pre or post immunization.

In the assay system, the uniquely designed multiple PCR primer set for each gene family are composed of specific Vβ, Dβ, and Jβ primers. The specificity of the developed technology has been validated by test on monoclonal T cell lines, polyclonal naïve cells of cord blood, and vaccine specific T cells. Monoclonal T cells, such as Jurkat cells (only express Vβ 8 monoclonal repertoire), show a single defined band, while T cells of the cord blood display a smear with no defined bands (see figure 1A). The specific clonal expansion with the HAV vaccine as neo-antigen results in the CD4+CD45RA+ subpopulation and can be detected using the assay system (see figure 1B). The accuracy and sensitivity of the assay system is comparable to a Genescan assay of the same sample. The system shows high reproducibility when repeating the test with the same samples.

The TCRExpress™ Clonality Detecting Kit was successfully used to monitor T cell immune response on the molecular level prior to and after HAV vaccine immunization in the CD4+ T cell subset of a human subject (Fig. 2). Figure 2A shows polyclonal expression (smear) in all displayed 22 Vβ families in the human subject, and one single band in the positive control from a monoclonal T cell line. In contrast to Panel A, there are in Panel B monoclonal expansions (Vβ 3, 4, 14, 20 and 23), oligoclonal expansions (Vβ 6, 8, 9, 17 and 21) and polyclonal expressions (all others) visible following the vaccination in the same subject.

Figure 2
 
Figure 2:TCRExpress™ Human TCR Vβ Repertoire Clonality Detecting Kit was used to successfully detect T cell immune response following vaccination with HAV vaccine. Panel A: Clonalities of 22 Vβ families (Vβ1 to Vβ24, except nonfunctional Vβ10 and 19) were detected using a.m. kit, which shows the polyclonal repertoire in a human CD4+ T cell subset prior to the HAV vaccine immunization. Panel B: In contrast to panel A, monoclonal Vβ families (Vβ3, 4, 14, 20 and 23) and oligoclonal Vβ6, 8, 9, 17 and 21) were detected using the same kit with CD4+ T cells of the same subject following vaccination with HAV vaccine. Column M shows the 10bp DNA size standard, column (+) the positive control from a monoclonal T cell line.

The bands of interest can be cut directly from the gel for sequencing of the CDR3 region with a DNA sequencer. We cut the smear band of the Vβ4 family from Fig. 2A as well as the single band of Vβ4 in Fig. 2B. The DNA was extracted from both gels and TA cloning performed. The sequencing data is shown below in Tab. 1 and 2. The smear band of Vβ4 (Tab. 1) shows polyclonal CDR3 sequences, while the single band of Vβ4 displays only one identical sequence (and only one Jβ sequence) (Tab. 2).

Table 1+2
 

To see examples for applications of clonality detection kits, please click here

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T cell receptor clonality/diversity of CDR3 determination and quantitative analysis kits

These kits are designed for the convenient, sensitive, reproducible and quantitative determination of changes in the diversity/clonality of the TCR Vα and Vβ repertoires in human and mouse T lymphocytes. The system can determine TCR α and β diversity/clonality in human and mouse T lymphocytes and their subsets, such as CD4, CD8, CD45RA and CD45RO T cell subsets of the CD4 and CD8 population, and CD4+CD25+ or CD4+CD25- subsets.

The kit contents include ready-to-use PCR amplification plates, proprietary PCR reaction buffers and also proprietary statistical analysis software. Kits with enzymes and RNA purification are available. The uniquely designed primers, essential reagents and control cDNA or RNA of monoclonal T cells have been pre-loaded in a 24-well PCR plate (32-well PCR plate for Human Vα) with chimney wells, which are more effective than flat-top plates in preventing contamination. For high resolution spectratyping and easy to perform molecular cloning, non-template nucleotides are added to the 5' end of the primers and all of the Vα/β primers are labeled with 6-FAM fluorescence. These additional nucleotides result in a high level of adenylation of the 3' end in the forward DNA strand to facilitate accurate spectratyping and TA cloning. The labeled Vα/β gene primers also are important factors for achieving high resolution spectratyping results.

Figure 3 shows the possibilities of TCR CDR3 diversity determination and quantitative analysis. The data shows two samples of the same subject, one before, and one after immunization. Prior to immunization all results are in an area of a Multiplication Factor (MF) < 3, meaning the results differ by less than +-3 times the Standard Deviation from the average value of the control samples. After immunization the picture significantly changes. Some values reach 45 times the standard deviation, therefore showing a significant change.

Figure 3
 
Figure 3: Quantitative analysis of TCR clonality/diversity successfully performed using TCRExpress clonality/diversity determination and quantitative analysis kit, used on the same samples as in Figure 2. The 2-dimensional figures show the degree of changes in the clonality/diversity for each gene family (upper panel). The 3-dimensional (3D) figures show not only the degree of change, but also their exact location (CDR3 length). MF - multiplication factor. An MF > 3 SD (Standard Deviation) is considered a significant change.

For analysis like the one above, it is important to generate a good foundation of control samples. This means the pure number of controls as well as their applicability. The number of controls should be > 5-10, and, for the above case for example, they should only come from non-immunized subjects.

To see examples for applications of the CDR3 diversity kits, please click here

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T cell receptor (TCR) Vα/β repertoire clone screening assay kits

These kits are designed for the sensitive, quick, and convenient screening of the TCR clone assay in the established monoclonal T cells, such as CTL clone, tumor T cell clone, or T cell line. The assay system can also be used to determine quantitatively the expression of TCR receptor genes when used with an appropriate gel analysis system, such as NIH-image. The kits can be used to screen in the TCR α or β chain within unfractionated T cells (PBMC) and T cell subsets, such as CD4, CD8, and CD45RA and CD45RO T cell subsets of CD4 and CD8 subsets of CD4 and CD8 populations in human and mouse subjects.

The clone screening system consists of two major components: PCR amplifications and the screening assay of particular TCR clones. The uniquely designed Vα/Vβ and internal control primer sets, essential reagents and control cDNA or RNA of monoclonal T cells have been preloaded in the 24-well PCR plates (32-well PCR plate for Human Vα) with chimney wells, which are more effective than flat-top plates in preventing contamination. Proprietary PCR reaction buffers and conditions play a critical role resulting in highly sensitive and specific multiple PCR amplifications. The Vβ assay system is based on the fact that the established monoclonal T cells express only one particular Vβ family, while polyclonal T cells express all Vβ families (see figure 4).

Figure 4
 
Figure 4: A TCRExpress™ T Cell Receptor Clone Screening Assay Kit was used to detect monoclonal Vβ 8 expression of the established monoclonal T cells (Panel A). Panel B shows the polyclonal expression of all Vβ repertoires in human PBMC.

In contrast to the Vβ chain, the Vα chain expresses two particular Vα families in the established clone cells (see figure 5). The most important and unique feature of the system is it simplicity, yielding rapid and reliable results. The system quickly and easily screens for the presence of a single TCR clone in established human or mouse monoclonal T cells using a easy to use gel analysis. Use of the kit does not require extensive training, and the expenses for testing are reduced. Moreover, any band of interest from a particular screened clone can be cut from the gel and purified for direct sequencing of the CDR3 region.

Figure 5
 
Figure 5: One step PCR amplification was performed in SupMclone plate I by adding the isolated total RNA from T lymphocytes. Nested PCR amplification was performed in SupMclone plate II by transferring PCR products from SupMclone plate I. Expression of 22 mouse Vα repertoires can be assessed and separated on 2% agarose gel (upper panel). Lower panel shows that the particular Vα 1 and 17 gene families can be detected on the agarose gel. M - 100 bp DNA marker; (+) - positive control (RNA was from mouse T cell line).

To see examples for applications of the clone screening kits, please click here

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Differences between TCRExpress™ & SuperTCRExpress

For our TCRExpress kits the user needs to prepare cDNA separately. Reagents to perform the necessary steps for preparation of cDNA are included in the kit, but the preparation adds an additional step in the process. Product variants are available that include reverse transcription (RT) reagents and Taq DNA polymerase (enzymes), which are necessary for the function of the kit. Customers that carry these enzymes can order the kits without them.

Our SuperTCRExpress kits work with RNA. Therefore no separate cDNA preparation is necessary. An RNA purification system is included. Product variants are available that include reverse transcription (RT) reagents and Taq DNA polymerase (enzymes), both of which are necessary for the function of the kit. Customers that carry these enzymes can order the kits without them.

Through the more compact process, SuperTCRExpress kits have an increased sensitivity, reduce the risk of contamination and are easier to handle than TCRExpress kits.

Figure 6
 
Figure 6: Differences between TCRExpress™ and SuperTCRExpress™ kits



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